Purification and characterization of an extracellular beta-lactamase produced by Acinetobacter calcoaceticus.

A beta-lactamase was purified 430-fold from the culture supernatant of Acinetobacter calcoaceticus by ion exchange chromatography on CM-Sephadex and affinity chromatography on phenylboronic-acid-agarose. The purified enzyme was homogeneous as judged by SDS-PAGE, and was characterized with respect to molecular mass (38 and 41 kDa by gel filtration on Sephadex G-75 and SDS-PAGE, respectively), pH optimum (pH 7.0), temperature optimum (45 degrees C) and isoelectric point (9.3). The beta-lactamase showed mainly cephalosporinase activity. It was inhibited by cloxacillin, carbenicillin, penicillanic acid sulphone (sulbactam) and aztreonam. It was not inhibited by clavulanic acid up to a concentration of 0.25 mM. Neither EDTA nor p-chlormercuribenzoate, up to concentrations of 1 or 100 mM, respectively, affected activity. According to these characteristics, it is a typical CEP-N cephalosporinase.

The alkaline phosphatase from bone: transphosphorylating activity and kinetic mechanism.

For the purified alkaline phosphatase from bone, the ability to catalyze a phosphate transfer reaction from p-nitrophenyl phosphate to two different hydroxy acceptor compounds, ethanolamine and glycerol, was established by identification of the formed phosphorylated products, phosphoethanolamine and glycerol 3-phosphate, respectively. In addition, a steady-state kinetic analysis of the hydrolysis of p-nitrophenyl phosphate in the presence of an added nucleophile, diethanolamine, gave rise to the proposal of a simple model for the kinetic mechanism of the enzyme. This mechanism includes a covalent phosphoryl enzyme intermediate, the dephosphorylation of which by water (k3) or a nucleophile (k4) is rate-determining. According to this model, in the presence of diethanolamine, k3 and k4 were determined to be 4.44 s-1 M-1 and 1000 s-1 M-1, respectively. Therefore, in vitro a suitable nucleophile, such as diethanolamine, seems to be a better phosphate acceptor than water. These results may suggest that alkaline phosphatase from bone could be well suited for catalyzing phosphate transfer reactions in vivo as well.

Lipopolysaccharide-protein interactions: determination of dissociation constants by affinity electrophoresis.

An affinity electrophoresis system is described to allow determination of dissociation constants of lipopolysaccharide (LPS)-protein complexes. The LPS ligand is incorporated into polyacrylamide gels by addition to the polyacrylamide-N,N'-methylenebisacrylamide polymerization mixture. Quantitative evaluation revealed formation of immobile protein-ligand complexes. The method was applied both to R- and S-form LPS from Acinetobacter calcoaceticus. For a heat-modifiable outer membrane protein with Mr 18,000 from strain 69V the dissociation constant was determined to be 0.5 mM (EDTA-salt extracted R-LPS) and 0.3 mM (phenol-chloroform-petrolether extracted R-LPS). In comparison, for another A. calcoaceticus strain, CCM 5593, a higher dissociation constant of 1.0 mM (phenol-chloroform-petrolether extracted R-LPS) -indicative of lower affinity - was obtained. When S-LPS from A. calcoaceticus 69V was incorporated into the affinity gels, a dissociation constant of 0.02 mM was determined which indicates much stronger interactions than those exerted by R-LPS forms.

Interactions between lipopolysaccharide and outer membrane proteins of Acinetobacter calcoaceticus studied by an affinity electrophoresis system.

R-Form lipopolysaccharides of Acinetobacter calcoaceticus could be incorporated into polyacrylamide gels in an immobile form by adding it directly to the acrylamide-N,N'-methylenebisacrylamide polymerization mixture. The separation of A. calcoaceticus 69 V outer membrane proteins in these affinity gels demonstrated a specific interaction with the lipopolysaccharide ligand for one of the proteins. This protein is heat-modifiable and has an Mr of about 18,000. By incorporation of varying concentrations of lipopolysaccharide, a dissociation constant of the protein-lipopolysaccharide complex of 0.5 mM could be determined. In comparison, for another A. calcoaceticus strain, CCM 5593, a higher dissociation constant (1.0 mM)--indicative of lower affinity--was obtained.

Effect of hexadecane-induced vesiculation on the outer membrane of Acinetobacter calcoaceticus.

Lipopolysaccharide-rich vesicles were released from Acinetobacter calcoaceticus 69V during growth on hexadecane. Vesicle formation occurred over the whole surface of the cell as demonstrated by scanning electron microscopy. In contrast, the surface of acetate-grown cells, for which little lipopolysaccharide was found in the growth medium, appeared smooth. The overall chemical composition as well as the protein and phospholipid composition of the outer membranes of both cell types was very similar. In the vesicles all outer membrane proteins were found with the exception of an Mr 10,000 polypeptide corresponding to Braun's lipoprotein. Compared with the outer membrane, the vesicles contained more phosphatidylethanolamine. Hexadecane-grown cells were susceptible to exogenously added phospholipase. Nevertheless the barrier function towards lysozyme was retained.

Production of an antiserum against cyclic nucleotide phosphodiesterase and its use for the immunocytochemical demonstration of this enzyme in rat cerebellum.

A procedure for the separation of cyclic AMP phosphodiesterase from a commercially available preparation and for raising antibodies against this enzyme in rabbits is described. An antiserum thus obtained was used for the immunocytochemical detection of cyclic nucleotide phosphodiesterase in rat cerebellum. The molecular layer, the granular layer and the cerebellar white matter exhibited different degrees of immunoreactivity. Only a few cell bodies (possibly glial cells) were stained. Most of the antigenic sites were present in the neuropil of the molecular layer and around Purkinje cells. Cerebellar glomeruli, sites of synaptic interactions between mossy fibres, Golgi cells and granule cells, were also stained by this antiserum. Control reactions using preimmune serum were consistently negative.

Effects of detergents and phospholipids on the pyridine nucleotide-independent aldehyde dehydrogenase from membranes of Acetobacter rancens.

The pyridine nucleotide-independent aldehyde dehydrogenase solubilized and purified from membranes of Acetobacter rancens CCM 1774 requires the presence of detergents for activity. While several detergents could stimulate the enzyme activity the stability of the enzyme-detergent complexes was rather low. Phospholipid substitution experiments revealed the reversibility of the loss of activity caused by phospholipid removal. Enzyme-phospholipid complexes generated from a complex phospholipid fraction exhibited an increased stability. The predominant effect of phosphatidylglycerol on both enzyme activity and stability is discussed in terms of essential boundary layer phospholipids.